Refolding Record:
| Protein | |
|---|---|
| Protein Name | Shiga toxin 1 B subunit |
| Abbreviated Name | STB |
| SCOP Family | Bacterial AB5 toxins, B-subunits |
| Structure Notes | |
| Organism | Escherichia coli |
| UniProt Accession | Q7WZI6 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | aa.21-89 |
| Chimera | Shiga toxin B:rotavisus nonstructural protein 4 fusion aa.86-175 |
| Variants | n/a |
| Chain Length | 200 |
| Molecular Weight | 23038.9 |
| Pi | 5.55 |
| Molecular Weight | 23038.9 |
| Disulphides | 1 |
| Full Sequence |
MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDRWGSE
TPDCVTGKVE YTKYNDDDTF TVKVGDKELF TNRWNLQSLL LSAQITGMTV TIKTNACHNG GGFSEVIFR
GPGP KEQIT TKDEIEKQMD RVVKEMRRQL EMIDKLTTRE IEQVELLKRI YDKLTVQTTG EIDMTKEINQ KNVRTLEEWE SGKNPYEPRE VTAAM
|
| Notes | Simian Rotavirus Nonstructural protein (NSP4) UniProt ID:O92323_9REOV, residues 86-175. Linker between the two proteins: Gly Pro Gly Pro |
| Expression | |
|---|---|
| Report | Choi NW, Estes MK, Langridge WH (2005) Vaccine, 23, 5168-76 |
| Project Aim | Vaccine studies |
| Fusion | N-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3)pLysS |
| Expression Temp | 37.0 |
| Expression Time | 3-4h |
| Expression Vector | pRSET-A |
| Expression Protocol | 250ml LB medium was inoculated with 25ml overnight culture and grown at 37degC until OD600 reached 0.5. IPTG was added to 1mM and cells were grown a further 3-4h, then harvested by centrifugation (4000g, 10min, 4degC) |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.5 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | n/a |
| Solubilization Buffer | 8M urea, 100mM NaCl, 20mM HEPES pH 8.0 |
| Refolding Buffer | 25mM Tris pH 8.3 |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | no |
| Refolding pH | 8.3 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Cells were lysed in 10ml solublization buffer by sonication on ice 3 times. The lysate was centrifuged (12000g, 10min, 4degC) and 10mM imidazole was added the supernatant, which was then loaded onto a 2ml Ni-NTA column. After washing, the protein was eluted from the column in solubilization buffer with 250mM imidazole. The protein was then dialyzed against refolding buffer. |
| Refolding Assay | Bioactivity,Receptor binding |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |